DNA Cloning Expands into New Horizons

Approaches Now Extend Implications of Technology Beyond Manipulation of Genes

The pJAZZ vectors were derived from phage N15 by deletion of the structural and lysis genes, followed by insertion of a multiple cloning site situated between a pair of transcriptional terminators (T). A drug-resistance gene was added to the right arm. Dark balls represent the hairpin telomeres; telN, protelomerase; repA, replication protein; cB, regulator of replication; CamR, chloramphenicol resistance. [Lucigen]

One of the most indispensable techniques in all areas of the life sciences is the use of expression vectors. Cloning a gene between two restriction sites, and the expression of a protein, are almost always taken for granted—but a frequently overlooked detail is that sometimes, as a promoter is placed upstream of a multiple cloning site, several base pairs, or even restriction sites, could be appended to the 5´-untranslated region of the gene of interest. These sequences are known to influence both prokaryotic and eukaryotic translation efficiency.

Hal S. Alper, Ph.D., assistant professor in the department of chemical engineering at the University of Texas at Austin, and colleagues, recently utilized a theoretical framework to predict the effect that a multiple cloning site has on translation efficiency, and the investigators subsequently applied this concept to redesign these sites and reduce variability associated with restriction site choice. “This is the first time that a performance-based analysis of multiple cloning sites has been conducted in yeast expression systems,” says Dr. Alper.

One of the most important ideas emerging from this work, recently published in Nucleic Acids Research, is that nucleotides placed between the promoter and the gene start site during cloning may shape translation efficiency, and this is a frequently overlooked aspect when using standardized expression vectors.

Moreover, the impact of the nucleotides is dependent on the promoter used, indicating an interaction in parts. “The idea in synthetic biology is building function from parts, and in some respects, the interactions between these individually characterized parts are often very context specific,” emphasizes Dr. Alper.

In addition to ensuring the successful expression of a protein of interest, this concept has even more profound implications. When the same gene is cloned between two different restriction sites within the same expression vector, the resulting phenotypes might be different because protein-expression levels are different.

“This concept, therefore, becomes important not only to optimize the expression of proteins from genes cloned into different restriction sites, but also for the ability to conduct genotype-phenotype associations,” explains Dr. Alper. Some of the new multiple cloning sites developed in this work are devoid of this site-to-site variation, which makes them functional and flexible multiple cloning sites for a variety of applications.

An issue of considerable concern in molecular biology is the manipulation of DNA fragments that are toxic or unstable, yet their successful cloning is essential to understand their biological function. “We recently developed a new system that enabled us to clone and express toxic genes that previously were unclonable in other expression systems,” says David Mead, Ph.D., president and CEO of Lucigen.

The expression vector pJAZZ is maintained as a linear plasmid. In addition, it contains transcriptional terminators on each side of the insert to minimize interference between the cloned genes and vector sequences.

Most notable of these unclonable sequences are repetitive DNA and highly AT-rich regions. Repetitive DNA is difficult to clone because repeats often recombine out of the vector. “In a circular vector, we have some evidence as to why repetitive regions are deleted. We believe that the torsional stress that exists in a circular plasmid causes the repetitive region to recombine out during replication and transcription,” explains Ronald Godiska, Ph.D., senior scientist at Lucigen.

This torsional stress is missing in the linear pJAZZ plasmid, and this ensures the stability of the repetitive DNA regions. “This is the most striking utility of the linear vector,” explains Dr. Godiska, lead author of a recent study that describes the research applications of this new cloning system. Illustrating the superiority of this system for applications that involve AT-rich regions, which are also unstable, Dr. Godiska and colleagues described the cloning and sequencing of the 69% AT-rich 3.1 Mb genome of the Flavobacterium columnare Gram-negative bacterium.

Another advantage of the linear cloning vector is that it eliminates the size bias. When a DNA fragment is incorporated into a circular vector, two ligation reactions occur—an intermolecular ligation that joins an end of the insert to the vector, followed by an intramolecular ligation that circularizes the molecule.

The efficiency of circularization decreases as the sizes of the vector or insert increase. In linear vectors, the cloning involves two independent ligation events, so this size bias is eliminated. “This represents another major advantage of the pJAZZ vector, and it allowed us to easily clone DNA fragments up to 20 kb without size bias,” explains Dr. Godiska.

“One of the things that we are trying to find out is whether we could use shorter homology regions between DNA fragments that undergo homologous recombination,” says Kumaran Narayanan, Ph.D., senior lecturer at the Monash University Sunway Campus, Malaysia, and adjunct assistant professor in the department of genetics and genomic sciences at Mount Sinai School of Medicine.

Homologous recombination in E. coli usually requires 40 to 50 base pairs of homology between the recombining DNA molecules to be detectable. “There is some evidence that 20 to 30 base pairs might be sufficient, and this would allow investigators to design shorter primers for recombination, facilitating more flexible changes to DNA and is more economical,” explains Dr. Narayanan.

Dr. Narayanan and colleagues recently optimized an E. coli homologous recombination system for the in vivo modification of DNA substrates, and further perfected it to allow the cloning of repetitive DNA regions, a form of DNA known to be inherently unstable in a recombination environment. This recombination protocol uses a 10–20 minute induction time to minimize DNA exposure to the recombination enzyme, and is ideal for functional studies that involve highly repetitive regions, trinucleotide repeats, and homologous genes.

An additional effort in Dr. Narayanan’s group focuses on generating linear chromosome vectors that have emerged as promising tools for gene delivery into cells. Dr. Narayanan and colleagues accomplished this by capping the ends of linearized DNA fragments with telomeres derived from bacteriophage N15 to provide protection from nuclease degradation and to enable DNA replication as a linear plasmid.

The investigators subsequently exemplified the strength of this technique by generating a linear 100 kb BAC that expressed the human β-globin gene in a human host cells.

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